Early stages of seed development in Brassica napus: a seed coat‐specific cysteine proteinase associated with programmed cell death of the inner integument

Abstract

SummaryA maternal plant exquisitely promotes the success of its offspring by orchestrating embryo development and endowing protection even after the embryos mature. It uses ovule integuments for physical and physiological contact with the developing embryo and for subsequently equipping the seed with a seed coat (testa). The testa is developmentally and metabolically dynamic, but its molecular biology is not well understood. We show here that the inner integument in Brassica napus undergoes organized development and then programmed cell death (PCD), as evident from vacuolation, starch mobilization, DNA fragmentation and eventual compression. We have identi®ed a cysteine proteinase gene (BnCysP1) that is expressed only in the inner integument as it undergoes PCD, well before the embryo begins storage protein synthesis. Two paralogous Cys proteinases have been recruited in rapeseed for the PCD of testa and for leaf senescence, and these differ 25% in their primary structure and post-translational modi®cations. Despite Arabidopsis being closely related to rapeseed, and an indication of developmental compression of its inner integument, the Arabidopsis genome is suggestive of only one Cys proteinase that shows »72% identity to BnCysP1. It is, however, leaf senescence-associated, and the other Cys proteinases are <52% identical. BnCysP1 also differs from ricinosome-deployed PCD Cys endopeptidases in lacking the hallmark KDEL tail and being glycosylated. BnCysP1, one of the very few plant genes known to function only in the seed coat, will be useful in dissecting post-fertilization development of this important organ in rapeseed.

Phosphorylation of the 12 S globulin cruciferin in wild-type and abi1-1 mutant Arabidopsis thaliana (thale cress) seeds

“…† Phosphorylation at Thr 8 or Ser 12 is not distinguishable in the present study. ‡ Phosphorylation at Tyr 14 or Tyr 20 is not distinguishable in the present study.…”

“…LC-MS/MS analysis identified a total of 19 phosphopeptides encompassing 20 phosphorylation sites in cruciferin, of which eight resided in the α-subunits and 11 in the β-subunits ( Table 1). The peptide ADVYKPQLGYISTLNSYDLPILR may be phosphorylated at either Tyr 10 or Ser 12 . MS/MS analysis was unable to confirm whether phosphorylation occurred at Thr 8 or Ser 12 in peptide VVPGCAETFMDSQPMQGQQQGQPWQGR, or at Tyr 14 or Tyr 20 in peptide CTENLDDPSDADVYKPSLGYISTLNSYN-LPILR.…”

“…Temporal and spatial factors influence the accumulation of seed storage proteins in the cotyledon and axis during seed development [10][11][12]. The latter is exemplified by the observation that antisense suppression of napin expression causes a corresponding increase in cruciferin synthesis, thus neutralizing the overall impact on total seed protein content [13].…”

Physiological and cytological aspects of Inga vera subsp. affinis embryos during storage

“…Because recalcitrant embryos are in a germination mode of development it seems plausible that they use starch to maintain their energy metabolism during moist storage and that low temperatures slow down metabolism. Starch mobilization has also been reported in the integument of early developing Brassica napus seeds, when it undergoes programmed cell death (Wan et al, 2002). Based on the SEM images, disappearance of starch granules correlated with the decline of viability during storage of I. vera embryos.…”

The classical Ubisch bodies carry a sporophytically produced structural protein (RAFTIN) that is essential for pollen development

“…Antiserum IgG was initially purified (24) and further purified with Affi-Gel 10 (Bio-Rad) following the suppliers' instructions. Plant protein extraction and Western blot analysis were performed essentially as described (25).…”